Multilayer electrochemical analyte sensors and methods for making and using them

ABSTRACT

Embodiments of the invention provide multilayer analyte sensors having material layers (e.g. high-density amine layers) and/or configurations of material layers that function to enhance sensor function, as well as methods for making and using such sensors. Typical embodiments of the invention include glucose sensors used in the management of diabetes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application that claims the benefitunder 35 U.S.C. 120 of U.S. patent application Ser. No. 15/891,264,filed Feb. 7, 2018, the contents of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to methods and materials useful foranalyte sensor systems, such as glucose sensors used in the managementof diabetes.

BACKGROUND OF THE INVENTION

Sensors are used to monitor a wide variety of compounds in variousenvironments, including in vivo analytes. The quantitative determinationof analytes in humans is of great importance in the diagnoses andmaintenance of a number of pathological conditions. Illustrativeanalytes that are commonly monitored in a large number of individualsinclude glucose, lactate, cholesterol, and bilirubin. The determinationof glucose concentrations in body fluids is of particular importance todiabetic individuals, individuals who must frequently check glucoselevels in their body fluids to regulate the glucose intake in theirdiets. The results of such tests can be crucial in determining what, ifany, insulin and/or other medication need to be administered.

Analyte sensors typically include components that convert interactionswith analytes into detectable signals that can be correlated with theconcentrations of the analyte. For example, some glucose sensors useamperometric means to monitor glucose in vivo. Such amperometric glucosesensors typically incorporate electrodes coated with glucose oxidase, anenzyme that catalyzes the reaction between glucose and oxygen to yieldgluconic acid and hydrogen peroxide (H₂O₂). The H₂O₂ formed in thisreaction alters an electrode current to form a detectable and measurablesignal. Based on the signal, the concentration of glucose in theindividual can then be measured.

A typical electrochemical glucose sensor works according to thefollowing chemical reactions:

The glucose oxidase is used to catalyze the reaction between glucose andoxygen to yield gluconic acid and hydrogen peroxide as shown inequation 1. The H₂O₂ reacts electrochemically as shown in equation 2,and the current is measured by a potentiostat. The stoichiometry of thereaction provides challenges to developing in vivo sensors. Inparticular, for optimal glucose oxidase based sensor performance, sensorsignal output should be determined only by the analyte of interest(glucose), and not by any co-substrates (O₂) or kinetically controlledparameters such as diffusion. If oxygen and glucose are present inequimolar concentrations, then the H₂O₂ is stoichiometrically related tothe amount of glucose that reacts with the glucose oxidase enzyme; andthe associated current that generates the sensor signal is proportionalto the amount of glucose that reacts with the enzyme. If, however, thereis insufficient oxygen for all of the glucose to react with the enzyme,then the current will be proportional to the oxygen concentration, notthe glucose concentration. Consequently, for a glucose sensor to providea signal that depends solely on the concentrations of glucose, glucosemust be the limiting reagent, i. e. the O₂ concentration must be inexcess for all potential glucose concentrations. A problem with usingsuch glucose sensors in vivo, however, is that the oxygen concentrationwhere the sensor is implanted in vivo is low relative to glucose, aphenomenon which can compromise the accuracy of glucose sensor readings(and consequently, this phenomenon is termed the “oxygen deficitproblem”).

Important components of certain electrochemical analyte sensors includethe layers of material that are disposed over the electrodes in orderallow the sensor to appropriately measure analyte signals in view ofissues such as the oxygen deficit problem discussed above. Based onvarious factors such as the material compositions used in layeredelectrochemical sensor stacks as well as where these compositions aredisposed within the stack architecture, a sensor may vary in terms ofits stability, reliability, and sensitivity in detecting analytesignals. In this context, there is a need in the art for multilayerelectrochemical sensors having layered materials that are optimized forsensor production and function. There is also a need for multilayerelectrochemical sensors having improved stability, reliability, andsensitivity in detecting analyte signals. Embodiments of the inventiondisclosed herein meet these as well as other needs.

SUMMARY OF THE INVENTION

Embodiments of the invention disclosed herein provide electrochemicalsensor designs that include multilayer analyte sensor stacks. In theseembodiments, the components of the multilayer analyte sensor stacks areformed from selected layers/materials and disposed within the stackarchitecture in a specific orientation that is designed to provide thesesensors with enhanced functional properties. The disclosure furtherprovides methods for making and using such sensors. As discussed indetail below, typical embodiments of the invention relate to the use ofa sensor that measures a concentration of an aqueous analyte of interestor a substance indicative of the concentration or presence of theanalyte in vivo (e.g. glucose sensors used in the management ofdiabetes). Embodiments of the invention provide innovative ways tosimplify the design of certain conventional electrochemical sensorshaving a plurality of layers disposed over electrodes.

An illustrative embodiment of the invention is an electrochemicalanalyte sensor comprising a base layer, a working electrode disposed onthe base layer, and a multilayer analyte sensor stack disposed upon theworking electrode. In this embodiment, the multilayer analyte sensorstack comprises an analyte sensing layer (e.g. one comprising glucoseoxidase) disposed directly on the working electrode, and this layerfunctions to detectably alter the electrical current at the workingelectrode in the presence of an analyte. In these embodiments, ahigh-density amine (“HDA”) layer which comprises polymers having aplurality of repeating amine groups (e.g. poly-l-lysine polymers) isdisposed directly on top of the analyte sensing layer. In theseembodiments, an analyte modulating layer (e.g. one comprising a glucoselimiting membrane which modulates the diffusion of glucose frominterstitial fluid to the working electrode) is further disposeddirectly on top of this high-density amine layer.

In typical analyte sensor embodiments, the multilayer analyte sensorstack does not comprise at least one of: a further layer comprising analbumin; a further layer comprising a siloxane adhesion promoting agent;or a layer comprising glutaraldehyde. For example, in the workingembodiments disclosed herein, the multilayer analyte sensor stackconsists essentially of the analyte sensing layer, the high-densityamine layer and the analyte modulating layer. In typical embodiments ofthe invention, the high-density amine layer comprises a first side indirect contact with the analyte sensing layer, and a second side indirect contact with the analyte modulating layer and this high densityamine layer functions as an adhesive layer that binds the analytesensing layer to the analyte modulating layer. Optionally, the analytesensing layer comprises glucose oxidase disposed in the layer so thatthe analyte sensor senses glucose; and the high-density amine layerfurther functions to decrease sensor signal changes that result fromfluctuating levels of oxygen (O₂). In illustrative working embodimentsof the invention disclosed herein, the polymers having a plurality ofrepeating amine groups that are used to form the high density aminelayer comprise poly-l-lysine polymers having molecular weights between30 KDa and 300 KDa, for example, molecular weights between 150 KDa and300 KDa. Typically, the polymers having a plurality of repeating aminegroups in the high-density amine layer are in amounts from 0.1weight-to-weight percent to 0.5 weight-to-weight percent. Optionally,the high-density amine layer is from 0.1 to 0.4 microns thick.

Another embodiment of the invention is a method of making anelectrochemical analyte sensor comprising the steps of: disposing aworking electrode on a base layer; disposing an analyte sensing layer(e.g. one comprising glucose oxidase) over the working electrode. Thesemethods further comprise disposing a high-density amine layer comprisingpolymers having a plurality of repeating amine groups (e.g.poly-l-lysine polymers) directly on the analyte sensing layer (e.g.using a spray or spin coating process); and disposing an analytemodulating layer (e.g. a glucose limiting membrane) directly on thehigh-density amine layer. Optionally, the electrochemical sensorcomprises a multilayer analyte sensor stack disposed over the workingelectrode, said multilayer analyte sensor stack consisting essentiallyof the analyte sensor layer, the high-density amine layer and theanalyte modulating layer. In the working embodiments disclosed herein,the analyte sensing layer comprises glucose oxidase disposed in thelayer so that the analyte sensor senses glucose; and the high-densityamine layer functions to decrease sensor signal changes that result fromfluctuating levels of oxygen (O₂) during glucose sensing in vivo.

Yet another embodiment of the invention is a method of sensing glucoseconcentrations in a fluid (e.g. the interstitial fluid of a diabeticpatient or another location where oxygen concentrations are low relativeto glucose), the method comprising disposing an electrochemical glucosesensor in the fluid, wherein the electrochemical glucose sensorcomprises a base layer; a working electrode disposed on the base layer;and a multilayer analyte sensor stack disposed on the working electrode.In these embodiments, the multilayer analyte sensor stack comprises ananalyte sensing layer comprising glucose oxidase disposed over theworking electrode that detectably alters the electrical current at theworking electrode in the presence of an glucose; a high-density aminelayer comprising polymers having a plurality of repeating amine groups(e.g. poly-l-lysine polymers), wherein the high-density amine layer isdisposed over the analyte sensing layer; and an analyte modulating layerdisposed over the high-density amine layer that comprises material themodulates the diffusion of glucose therethrough. These methods furthercomprise monitoring fluctuations in electrical conductivity that occurin the present of glucose; and correlating the fluctuations inelectrical conductivity with a concentration of glucose so that glucoseconcentrations in the fluid are sensed. In such embodiments, thehigh-density amine layer functions to increase adhesion between thelayers of the multilayer analyte sensor stack while simultaneouslydecreasing sensor signal changes that result from fluctuating levels ofoxygen (O₂) as glucose concentrations in the fluid are sensed.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic that illustrates the general structure ofhigh-density amine (HDA) polymer units that can be used to makehigh-density amine polymers. R1 comprises alkyl functional groups, forexample those comprising between 1-20 carbon atoms. R2 comprises ketonefunctional groups, for example those comprising at least one oxygen atomand between 1-20 carbon atoms. R3 comprises nitrogen functional groups,for example those comprising at least one nitrogen atom and between 1-20carbon atoms.

FIG. 2A provides a schematic showing a conventional (PRIOR ART) sensordesign comprising an amperometric analyte sensor formed from a pluralityof planar layered elements which include albumin protein layer and anadhesion promoter layer. FIG. 2B provides a schematic showingdifferences between such conventional multilayer sensor stacks and thenovel sensor stacks that are disclosed herein (i.e. sensor stacks thatdo not comprise a layer that includes glutaraldehyde, a layer thatincludes serum albumin, or a layer that includes a siloxane adhesionpromoter).

FIG. 3 provides a perspective view illustrating a subcutaneous sensorinsertion set, a telemetered characteristic monitor transmitter device,and a data receiving device embodying features of the invention.

FIG. 4 shows a schematic of a potentiostat that may be used to measurecurrent in embodiments of the present invention. As shown in FIG. 4, apotentiostat 300 may include an op amp 310 that is connected in anelectrical circuit so as to have two inputs: Vset and Vmeasured. Asshown, Vmeasured is the measured value of the voltage between areference electrode and a working electrode. Vset, on the other hand, isthe optimally desired voltage across the working and referenceelectrodes. The current between the counter and reference electrode ismeasured, creating a current measurement (isig) that is output from thepotentiostat.

FIG. 5 shows data from an in vivo study monitoring glucose in pigs usinga amperometric glucose sensor comprising an HDA material layer that hasbeen ethylene oxide (ETO) sterilized. This data shows that, followingETO sterilization, glucose sensor comprising an HDA material layerexhibit excellent ability to sense glucose in-vivo sensor over at least11 days of wear.

FIG. 6 is a graph of data from sensor embodiments of HDA multilayerstacks formed from poly-l-lysine having different molecular weights.This data confirms that sensor embodiments having HDA multilayer stackshave lower baseline oxygen responses as compared to sensor embodimentsformed using conventional multilayer stacks.

FIG. 7 shows data from an in vivo comparative study monitoring glucosein pigs using a amperometric glucose sensor comprising an HDA materiallayer as disclosed herein and compared to conventional sensors nothaving an HDA layer (e.g. FIG. 2A). Data from this study shows toeffectiveness of glucose sensors having HDA polymer layers that, forexample, function as adhesion promoters etc. in electrochemical analytesensors having multilayer sensor stacks.

FIG. 8 shows data from an in vitro comparative study monitoring glucosesensing under different concentrations of O_(in) order to compare theoxygen response in legacy/conventional glucose oxidase based sensorswith amperometric glucose sensor comprising an HDA material layer asdisclosed herein. These studies show that glucose sensors comprising anHDA material layer as disclosed herein display lower in-vitro signalchanges to oxygen concentration changes as compared tolegacy/conventional glucose oxidase based sensors.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations, and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings may be defined herein for clarity and/or for ready reference,and the inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. A number of termsare defined below.

All numbers recited in the specification and associated claims thatrefer to values that can be numerically characterized with a value otherthan a whole number (e.g. the diameter of a circular disc) areunderstood to be modified by the term “about”. Where a range of valuesis provided, it is understood that each intervening value, to the tenthof the unit of the lower limit unless the context clearly dictatesotherwise, between the upper and lower limit of that range and any otherstated or intervening value in that stated range, is encompassed withinthe invention. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges, and are alsoencompassed within the invention, subject to any specifically excludedlimit in the stated range. Where the stated range includes one or bothof the limits, ranges excluding either or both of those included limitsare also included in the invention. Furthermore, all publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. Publications cited herein are cited for theirdisclosure prior to the filing date of the present application. Nothinghere is to be construed as an admission that the inventors are notentitled to antedate the publications by virtue of an earlier prioritydate or prior date of invention. Further the actual publication datesmay be different from those shown and require independent verification.

The term “analyte” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a substanceor chemical constituent in a fluid such as a biological fluid (forexample, blood, interstitial fluid, cerebral spinal fluid, lymph fluidor urine) that can be analyzed. Analytes can include naturally occurringsubstances, artificial substances, metabolites, and/or reactionproducts. In common embodiments, the analyte is glucose. However,embodiments of the invention can be used with sensors designed fordetecting a wide variety other analytes. Illustrative analytes includebut are not limited to, lactate as well as salts, sugars, proteins fats,vitamins and hormones that naturally occur in vivo (e.g. in blood orinterstitial fluids). The analyte can be naturally present in thebiological fluid or endogenous; for example, a metabolic product, ahormone, an antigen, an antibody, and the like. Alternatively, theanalyte can be introduced into the body or exogenous, for example, acontrast agent for imaging, a radioisotope, a chemical agent, afluorocarbon-based synthetic blood, or a drug or pharmaceuticalcomposition, including but not limited to insulin. The metabolicproducts of drugs and pharmaceutical compositions are also contemplatedanalytes.

The term “sensor” for example in “analyte sensor,” is used in itsordinary sense, including, without limitation, means used to detect acompound such as an analyte. A “sensor system” includes, for example,elements, structures and architectures (e.g. specific 3-dimensionalconstellations of elements) designed to facilitate sensor use andfunction. Sensor systems can include, for example, compositions such asa layer of material having selected properties such as a high densityamine layer formed from polymers having a plurality of repeating aminegroups (e.g. a high-density amine layer comprising from 0.1weight-to-weight percent to 0.5 weight-to-weight percent poly-l-lysinehaving molecular weights between 150 KDa and 300 KDa), as well aselectronic components such as elements and devices used in signaldetection and analysis (e.g. current detectors, monitors, processors andthe like).

The terms “electrochemically reactive surface” and “electroactivesurface” as used herein are broad terms and are used in their ordinarysense, including, without limitation, the surface of an electrode wherean electrochemical reaction takes place. In one example, a workingelectrode measures hydrogen peroxide produced by the enzyme catalyzedreaction of the analyte being detected, creating an electric current(for example, detection of glucose analyte utilizing glucose oxidaseproduces H₂O₂ as a byproduct, H₂O₂ reacts with the surface of theworking electrode producing two protons (2H⁺), two electrons (2e⁻) andone molecule of oxygen (O₎ which produces the electronic current beingdetected). In the case of the counter electrode, a reducible species,for example, O₂ is reduced at the electrode surface in order to balancethe current being generated by the working electrode.

As discussed in detail below, embodiments of the invention relate to theuse of an electrochemical sensor that measures a concentration of ananalyte of interest or a substance indicative of the concentration orpresence of the analyte in fluid. In some embodiments, the sensor is acontinuous device, for example a subcutaneous, transdermal, orintravascular device. In some embodiments, the device can analyze aplurality of intermittent blood samples. The sensor embodimentsdisclosed herein can use any known method, including invasive, minimallyinvasive, and non-invasive sensing techniques, to provide an outputsignal indicative of the concentration of the analyte of interest. Theproduct is then measured using electrochemical methods and thus theoutput of an electrode system functions as a measure of the analyte.

Embodiments of the invention disclosed herein provide sensors of thetype used, for example, in subcutaneous or transcutaneous monitoring ofblood glucose levels in a diabetic patient. A variety of implantable,electrochemical biosensors have been developed for the treatment ofdiabetes and other life-threatening diseases. Many existing sensordesigns use some form of immobilized enzyme to achieve theirbio-specificity. Embodiments of the invention described herein can beadapted and implemented with a wide variety of known electrochemicalsensors, including for example, U.S. Patent Application No. 20050115832,U.S. Pat. Nos. 6,001,067, 6,702,857, 6,212,416, 6,119,028, 6,400,974,6,595,919, 6,141,573, 6,122,536, 6,512,939 5,605,152, 4,431,004,4,703,756, 6,514,718, 5,985,129, 5,390,691, 5,391, 250, 5,482,473,5,299,571, 5,568,806, 5,494,562, 6,120,676, 6,542,765, 7,033,336 as wellas PCT International Publication Numbers WO 01/58348, WO 04/021877, WO03/034902, WO 03/035117, WO 03/035891, WO 03/023388, WO 03/022128, WO03/022352, WO 03/023708, WO 03/036255, WO03/036310 WO 08/042,625, and WO03/074107, and European Patent Application EP 1153571, the contents ofeach of which are incorporated herein by reference.

Illustrative Embodiments of the Invention and Associated Characteristics

Embodiments of the invention disclosed herein provide sensors designedto include multilayer analyte sensor stacks formed from selectedmaterials that provide the sensors with enhanced functional and/ormaterial properties. The disclosure further provides methods for makingand using such sensors. As discussed in detail below, typicalembodiments of the invention relate to the use of a sensor that measuresa concentration of an aqueous analyte of interest or a substanceindicative of the concentration or presence of the analyte in vivo. Insome embodiments, the sensor is a subcutaneous, intramuscular,intraperitoneal, intravascular or transdermal device. Typically, thesensor can be used for continuous analyte monitoring. The sensorembodiments disclosed herein can use any known method, includinginvasive, minimally invasive, and non-invasive sensing techniques, toprovide an output signal indicative of the concentration of the analyteof interest. Embodiments of the invention provide an innovative way tosimplify the design of conventional electrochemical sensors having aplurality of layers disposed over the working electrode.

Embodiments of the invention having a constellation of elementsincluding a high density amine layer exhibit a number of advantages overconventional multilayer electrochemical sensor designs (e.g. asdisclosed in FIG. 2A). For example, embodiments of the invention havefewer layers of materials, a property that can be used to simplify themanufacturing process & reduce sensor-to-sensor variation as compared toconventional processes for making analyte sensor stacks, processes whichutilize a multicomponent adhesive layer to “glue” GLM to GOx (where alayer is created in-situ through many simultaneous chemical reactions,resulting in sensor-to-sensor variability). See FIG. 2B for a comparisonof conventional analyte sensors and the HDA sensors disclosed herein.Another advantage is that certain HDA sensor embodiments disclosedherein do not comprise Human Serum Albumin (HSA). Other advantages ofembodiments of the invention include improvements in stability that comewith the elimination of glutaraldehyde (glutaraldehyde cross-linkedglucose oxidase in conventional sensor designs may decrease GOx activityand stability). By removing glutaraldehyde, we remove this potentialcause of sensor instability. Another associated advantage is that thesensor embodiments disclosed herein are observed to exhibit more robuststerilization profile in e-beam and ETO processes.

Other associated advantages of the unique constellations of layeredmaterials disposed over working electrode(s) that are disclosed hereininclude more robust layer surfaces and increased stability over arrangeof different initialization profiles (sensors comprising HDA layers arestable under a range of different initialization profiles). Otheradvantages can include, for example, a more uniform layers as well asmore sites for electrochemical reactions, features which contribute tothe stability and/or sensitivity of layered sensor structures. Forexample, by providing smoother and more adhesive surfaces that cancontribute to sensor stability by decreasing the possibility that one ormore layers of material may delaminate. Importantly, a key advantage isan improved oxygen response that is observed in glucose oxidase basedsensors formed with HDA layer, with HDA Poly-l-lysine sensors showingless signal changes over variable oxygen concentrations (5% to 1%). Thisproperty addresses the oxygen deficit problem with glucose sensors thatis discussed above (as illustrated in data from illustrative workingexamples of HDA comprising sensor embodiments shown in FIGS. 6 and 8).FIG. 7 then shows data from an in vivo comparative study monitoringglucose in pigs using a amperometric glucose sensor comprising an HDAmaterial layer as disclosed herein and compared to conventional sensorsnot having an HDA layer (e.g. FIG. 2A).

In the high density amine layers disclosed herein, the polymers having aplurality of repeating amine groups can adopt a variety ofconfigurations. The simplest polymer architecture having a plurality ofrepeating amine groups is a linear chain: a single backbone with nobranches. Alternatively, the polymer can be branched. A branched polymermolecule is composed of a main chain with one or more substituent sidechains or branches. Special types of branched polymers includedendrimers. Dendrimers are a special case of macromolecules whereinevery monomer unit is branched. In some embodiments of the invention,the polymers having a plurality of repeating amine groups within the HDAlayer exhibit linear, and/or branched and/or dendrimer like structures.In illustrative embodiments of the invention disclosed herein, the HDAlayer comprises poly-l-lysine polymers.

While the illustrative working embodiments of the invention are formedfrom linear polymers, the polymers having a plurality of repeating aminegroups within the HDA layer can exhibit linear, branched and/ordendrimer like structures. Such HDA polymers include, for example,Poly-l-lysine, Poly-D-lysine, Chitosan, Amino-dextran, Polyethyleneimine, other Poly-l-amino acid polymers and the like. In certainembodiments of the invention, polymers comprise the general structureshown below with R1, R2 and R3 where:

-   -   R¹=Alkyl functional groups of various chain lengths (linear        and/or brand    -   R²=Ketone functional group    -   R³=Nitrogen functional group        In certain embodiments, the polymer comprises a poly-l-lysine        unit:

In one specific illustrative embodiment, the polymer comprises amolecular structure such as:

In another specific illustrative embodiment, the polymer comprises amolecular structure such as:

The high-density amine layer can be formed according to art acceptedprocesses, for example by weighing out applicable amount of a polymersuch as poly-l-lysine, dissolving this amount of poly-l-lysine inapplicable amount of water so that a clear solution is formed andstirring for 1 hour. Artisans can then use this solution to make aconcentration of 0.1 to 0.5 weight-to-weight percent (w/w %)high-density amine composition to form a layer in a sensor disclosedherein. Typically, the layer is applied to the senor stack by sprayingthe poly-l-lysine solution onto the substrate some number of times (e.g.3X wherein the biodot repeats a spray cycle), so that more repeatedapplications=more material deposited onto the substrates. In workingembodiments disclosed herein, poly-l-lysine polymers having differentmolecular weights were examiner, with HMW=High Molecular Weight=150 to300 KDa, MMW=Medium Molecular Weight=70 to 150 KDa, and LMW=LowMolecular Weight=30 to 70 KDa.

An illustrative embodiment of the invention is an electrochemicalanalyte sensor comprising a base layer, a working electrode disposed onthe base layer, and a multilayer analyte sensor stack disposed upon theworking electrode. In this embodiment, the multilayer analyte sensorstack comprises an analyte sensing layer disposed directly on theworking electrode, wherein the analyte sensing layer detectably altersthe electrical current at the working electrode in the presence of ananalyte, a high-density amine layer disposed over the analyte sensinglayer, wherein the high-density amine layer comprises poly-l-lysinepolymers, and an analyte modulating layer (e.g. a glucose limitingmembrane) disposed over the high-density amine layer, wherein theanalyte modulating layer modulates the diffusion of analyte (e.g.glucose) from an external environment (e.g. interstitial fluid) to theworking electrode.

In such analyte sensor embodiments, the multilayer analyte sensor stackdoes not comprise at least one of: a further layer comprising an albumin(and optionally no sensor layer comprises an albumin); a further layercomprising a siloxane adhesion promoting agent; or a further layercomprising glutaraldehyde (and optionally no sensor layer is formedusing glutaraldehyde or comprises glutaraldehyde moieties). For example,in the working embodiments disclosed herein, the multilayer analytesensor stack consists essentially of the analyte sensing layer, thehigh-density amine layer and the analyte modulating layer. In typicalembodiments of the invention, the high-density amine layer comprises afirst side in direct contact with the analyte sensing layer, and asecond side in direct contact with the analyte modulating layer, contactwhich allows this layer to function as an adhesive layer that binds theanalyte sensing layer to the analyte modulating layer. Optionally, theanalyte sensing layer comprises glucose oxidase disposed in the layer sothat the analyte sensor senses glucose; and the high-density amine layerfurther functions to decrease sensor signal changes that result fromfluctuating levels of O₍see, e.g. the data from illustrative embodimentsof the invention shown in FIG. 6). The polymers having a plurality ofrepeating amine groups within the HDA layer exhibit linear, and/orbranched and/or dendrimer like structures. In certain embodiments of theinvention, the poly-l-lysine in the high-density amine layer hasmolecular weights between 30 KDa and 300 KDa, for example, molecularweights between 150 KDa and 300 KDa. Typically, the poly-l-lysine in thehigh-density amine layer is in amounts from 0.1 weight-to-weight percentto 0.5 weight-to-weight percent. Optionally, the high-density aminelayer is from 0.1 to 0.4 microns thick. These 0.1 to 0.4 micron thinadhesive layers have unexpected advantages in that they exhibit a loweroxygen response as well as faster hydration times as compared toconventional sensors not having such thin HDA layers.

Another embodiment of the invention is a method of making anelectrochemical analyte sensor comprising the steps of: disposing aworking electrode on a base layer; disposing an analyte sensing layerover the working electrode, wherein the analyte sensing layer detectablyalters the electrical current at the working electrode in the presenceof an analyte; disposing a high-density amine layer comprising forexample HDA polymers directly on the analyte sensing layer (e.g. using aspray coating process); and disposing an analyte modulating layerdirectly on the high-density amine layer, wherein the analyte modulatinglayer modulates the diffusion of analyte therethrough so that anelectrochemical analyte sensor is made. Optionally, the electrochemicalsensor comprises a multilayer analyte sensor stack disposed over theworking electrode, said multilayer analyte sensor stack consistingessentially of the analyte sensor layer, the high-density amine layerand the analyte modulating layer. An illustrative poly-l-lysine solutionused to make an embodiment of the invention is 150 to 300 KDapoly-l-lysine, 0.3 poly-l-lysine w/w %, that is applied in approximately2 spray repeats. In the working embodiments disclosed herein, theanalyte sensing layer comprises glucose oxidase disposed in the layer sothat the analyte sensor senses glucose; and the high-density amine layerfunctions to decrease sensor signal changes that result from fluctuatinglevels of oxygen (O₂) during glucose sensing.

One unexpected advantage of analyte sensors comprising the high-densityamine layers disclosed herein is their ability to maintain excellentfunctionality (i.e. analyte sensing) following sterilization by EthyleneOxide. Specifically, when devices such as analyte sensors are sterilizedwith ethylene oxide, problems can arise if the ethylene oxide reactswith, and inhibits the activity of one or more sensitive components ofthe device, such as the enzyme glucose oxidase in amperometric glucosesensors. Such problems can prevent the effective use of ethylene oxidesterilization procedures on such devices. Methods and materials designedto address such challenges in this technology (e.g. the high-densityamine layers disclosed herein) are therefore desirable. In this context,embodiments of the invention include methods for making analyte sensorscomprising the high-density amine layers disclosed herein which includethe step of sterilizing the sensor with ethylene oxide, sensorscomprising the high-density amine layers disclosed herein that have beensterilized with ethylene oxide, and methods for sensing analytes such asglucose with these sensors comprising the high-density amine layersdisclosed herein that have been sterilized with ethylene oxide. Avariety of ethylene oxide sterilization procedures are known in the art(see, e.g. U.S. Patent Publications 20120252125, 20110233068,20070292305 and 20050089442, the contents of which are incorporatedherein by reference). Illustrative ethylene oxide parameters are asfollows.

Parameter Level Units Ethylene Oxide 200-800 mg/L Concentration Humidity >=30 % RH Temperature <=140 F Dwell Time  2-12 Hours CO2 mixture  <=80%% compositionFIG. 5 shows data from an in vivo study monitoring glucose in pigs usinga amperometric glucose sensor comprising an HDA material layer that hasbeen ethylene oxide (ETO) sterilized following such ETO sterilizationparameters. This data shows that, following ETO sterilization, glucosesensor comprising an HDA material layer exhibit excellent ability tosense glucose in-vivo sensor over at least 11 days of wear.

Yet another embodiment of the invention is a method of sensing glucoseconcentrations in a fluid (e.g. an environment where the concentrationsof glucose are low relative to the concentrations of oxygen) comprisingdisposing an electrochemical glucose sensor in the fluid, wherein theelectrochemical glucose sensor comprises a base layer; a workingelectrode disposed on the base layer; and a multilayer analyte sensorstack disposed on the working electrode. In these embodiments, themultilayer analyte sensor stack comprises an analyte sensing layercomprising glucose oxidase disposed over the working electrode, whereinthe analyte sensing layer detectably alters the electrical current atthe working electrode in the presence of an analyte. This stack alsoincludes a high-density amine layer, for example one comprisingpoly-l-lysine polymers, wherein the high-density amine layer is disposedover the analyte sensing layer; and an analyte modulating layer disposedover this high-density amine layer, wherein the analyte modulating layermodulates the diffusion of glucose therethrough. These methods furthercomprise monitoring fluctuations in electrical conductivity that can beobserved when glucose reacts with glucose oxidase; and correlating thefluctuations in electrical conductivity with a concentration of glucoseso that glucose concentrations in the fluid are sensed. In suchembodiments, the high-density amine layer functions to increase adhesionbetween the layers of the multilayer analyte sensor stack whilesimultaneously decreasing sensor signal changes that result fromfluctuating levels of oxygen (O₂) as glucose concentrations in the fluidare sensed. Optionally, the analyte modulating layer is a glucoselimiting membrane that comprises a polyurethane/polyurea polymer formedfrom a mixture comprising a diisocyanate, a hydrophilic polymercomprising a hydrophilic diol or hydrophilic diamine, and a siloxanehaving an amino, hydroxyl or carboxylic acid functional group at aterminus.

In typical embodiments of the invention, electrochemical sensors areoperatively coupled to a sensor input capable of receiving signals fromthe electrochemical sensor; and a processor coupled to the sensor input,wherein the processor is capable of characterizing one or more signalsreceived from the electrochemical sensor. In certain embodiments of theinvention, the electrical conduit of the electrode is coupled to apotentiostat (see, e.g. FIG. 4). Optionally, a pulsed voltage is used toobtain a signal from an electrode. In typical embodiments of theinvention, the processor is capable of comparing a first signal receivedfrom a working electrode in response to a first working potential with asecond signal received from a working electrode in response to a secondworking potential. Optionally, the electrode is coupled to a processoradapted to convert data obtained from observing fluctuations inelectrical current from a first format into a second format. Suchembodiments include, for example, processors designed to convert asensor current Input Signal (e.g. ISIG measured in nA) to a bloodglucose concentration.

In many embodiments of the invention, the sensors comprise abiocompatible region adapted to be implanted in vivo. In someembodiments, the sensor comprises a discreet probe that pierces an invivo environment. In embodiments of the invention, the biocompatibleregion can comprise a polymer that contacts an in vivo tissue.Optionally, the polymer is a hydrophilic polymer (e.g. one that absorbswater). In this way, sensors used in the systems of the invention can beused to sense a wide variety of analytes in different aqueousenvironments. In some embodiments of the invention, the electrode iscoupled to a piercing member (e.g. a needle) adapted to be implanted invivo. While sensor embodiments of the invention can comprise one or twopiercing members, optionally such sensor apparatuses can include 3 or 4or 5 or more piercing members that are coupled to and extend from a baseelement and are operatively coupled to 3 or 4 or 5 or moreelectrochemical sensors (e.g. microneedle arrays, embodiments of whichare disclosed for example in U.S. Pat. Nos. 7,291,497 and 7,027,478, andU.S. patent Application No. 20080015494, the contents of which areincorporated by reference).

In some embodiments of the invention, the apparatus comprises one ormore working electrodes, counter electrodes and reference electrodes,optionally clustered together in units consisting essentially of oneworking electrode, one counter electrode and one reference electrode;and the clustered units are longitudinally distributed on the base layerin a repeating pattern of units. In some sensor embodiments, thedistributed electrodes are organized/disposed within a flex-circuitassembly (i.e. a circuitry assembly that utilizes flexible rather thanrigid materials). Such flex-circuit assembly embodiments provide aninterconnected assembly of elements (e.g. electrodes, electricalconduits, contact pads and the like) configured to facilitate wearercomfort (for example by reducing pad stiffness and wearer discomfort).

As noted above, the sensor electrodes of the invention are coated with aplurality of materials having properties that, for example, facilitateanalyte sensing. In typical embodiments of the invention, an analytesensing layer is disposed directly on a working electrode, and includesan agent that is selected for its ability to detectably alter theelectrical current at the working electrode in the presence of ananalyte. In the working embodiments of the invention that are disclosedherein, the agent is glucose oxidase, a protein that undergoes achemical reaction in the presence of glucose that results in analteration in the electrical current at the working electrode. Theseworking embodiments further include an analyte modulating layer disposedover the analyte sensing layer, wherein the analyte modulating layermodulates the diffusion of glucose as it migrates from an in vivoenvironment to the analyte sensing layer. In certain embodiments of theinvention, the analyte modulating layer comprises a hydrophiliccomb-copolymer having a central chain and a plurality of side chainscoupled to the central chain, wherein at least one side chain comprisesa silicone moiety. In certain embodiments of the invention, the analytemodulating layer comprises a blended mixture of: a linearpolyurethane/polyurea polymer, and a branched acrylate polymer; and thelinear polyurethane/polyurea polymer and the branched acrylate polymerare blended at a ratio of between 1:1 and 1:20 (e.g. 1:2) by weight %.Typically, this analyte modulating layer composition comprises a firstpolymer formed from a mixture comprising a diisocyanate; at least onehydrophilic diol or hydrophilic diamine; and a siloxane; that is blendedwith a second polymer formed from a mixture comprising: a2-(dimethylamino)ethyl methacrylate; a methyl methacrylate; apolydimethyl siloxane monomethacryloxypropyl; a poly(ethylene oxide)methyl ether methacrylate; and a 2-hydroxyethyl methacrylate. Asdisclosed herein, additional material layers can be included in suchapparatuses. For example, in typical embodiments of the invention, theapparatus comprises a high-density amine layer which is disposed betweenand in direct contact with the analyte sensing layer and the analytemodulating layer so as to exhibit a number of beneficial propertiesincluding an ability to provide a smoother surface structure and furtherpromote adhesion between the analyte sensing layer and the analytemodulating layer. Without being bound by a specific scientific theory ormechanism of action, it is believed that adhesion between layers ispromoted by smoother layer contact architectures as well as Vander Waalsforce interactions between the HDA polymers in the HDA layer andcompounds present in the analyte sensing layer that is disposed on afirst side of this HDA layer, and Vander Waals force interactionsbetween the HDA polymers and compounds present in the analyte modulatinglayer that is disposed on a second side of this HDA layer (i.e. so thatthe HDA layer is in a “sandwich” configuration).

One prior art conventional sensor embodiment shown in FIG. 2A is aamperometric sensor 100 having a plurality of layered elements includinga base layer 102, a conductive layer 104 (e.g. one comprising theplurality of electrically conductive members) which is disposed onand/or combined with the base layer 102. The following comments relateto this conventional sensor which is described to help understand thedifferences between such conventional sensors and the inventiondisclosed herein. Typically, the conductive layer 104 comprises one ormore electrodes. An analyte sensing layer 110 (typically comprising anenzyme such as glucose oxidase) is disposed on one or more of theexposed electrodes of the conductive layer 104. A protein layer 116disposed upon the analyte sensing layer 110. An analyte modulating layer112 is disposed above the analyte sensing layer 110 to regulate analyte(e.g. glucose) access with the analyte sensing layer 110. An adhesionpromoter layer 114 is disposed between layers such as the analytemodulating layer 112 and the analyte sensing layer 110 as shown in FIG.2A in order to facilitate their contact and/or adhesion. This embodimentalso comprises a cover layer 106 such as a polymer coating can bedisposed on portions of the sensor 100. Apertures 108 can be formed inone or more layers of such sensors. Amperometric glucose sensors havingthis type of design are disclosed, for example, in U.S. PatentApplication Publication Nos. 20070227907, 20100025238, 20110319734 and20110152654, the contents of each of which are incorporated herein byreference. FIG. 2B shows a comparison between these conventionalmultilayer sensor stacks and the invention disclosed herein (i.e. onescomprising a HDA layer 500).

As noted above, embodiments of the invention also include methods formaking and using the HDA multilayer sensor stacks disclosed herein. Yetanother embodiment of the invention is a method of sensing an analytewithin the body of a mammal. Typically, this method comprises implantingan analyte sensor having an HDA multilayer sensor stack within themammal (e.g. in the interstitial space of a diabetic individual),sensing an alteration in current at the working electrode in thepresence of the analyte; and then correlating the alteration in currentwith the presence of the analyte, so that the analyte is sensed.

Embodiments of the invention also provide articles of manufacture andkits for observing a concentration of an analyte. In an illustrativeembodiment, the kit includes a sensor comprising a HDA multilayer sensorstack as discussed herein. In typical embodiments, the sensors aredisposed in the kit within a sealed sterile dry package. Optionally thekit comprises an insertion device that facilitates insertion of thesensor. The kit and/or sensor set typically comprises a container, alabel and an analyte sensor as described above. Suitable containersinclude, for example, an easy to open package made from a material suchas a metal foil, bottles, vials, syringes, and test tubes. Thecontainers may be formed from a variety of materials such as metals(e.g. foils) paper products, glass or plastic. The label on, orassociated with, the container indicates that the sensor is used forassaying the analyte of choice. The kit and/or sensor set may includeother materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes, and packageinserts with instructions for use.

Specific aspects of embodiments of the invention are discussed in detailin the following sections.

Typical Elements, Configurations and Analyte Sensor Embodiments of theInvention A. Typical Elements Found in of Embodiments of the Invention

The invention disclosed herein includes compositions comprisinghigh-density amine (HDA) polymers, compositions which can be used inlayered electrochemical sensor stacks as a way to impart functionalbenefits to the sensors. FIG. 1 provides a schematic that illustratesthe general structures of such polymers can be used to make thesepolymers.

FIG. 2A illustrates a cross-section of a conventional sensor embodiment100. The components of the sensor are typically characterized herein aslayers in this layered electrochemical sensor stack because, forexample, it allows for a facile characterization of conventional sensorstructures such as those shown in FIG. 2A and their differences from theinvention disclosed herein as shown in FIG. 2B (i.e. ones comprising aHDA layer 500). Artisans will understand, that in certain embodiments ofthe invention, the sensor constituents are combined such that multipleconstituents form one or more heterogeneous layers. In this context,those of skill in the art understand that, while certainlayers/components of conventional sensor embodiments are useful in theHDA sensors disclosed herein, the placement and composition of thelayered constituents is very different in HDA sensor embodiments of theinvention. Those of skill in this art will understand that certainembodiments if the invention include elements/layers that are found inconventional sensors while others are excluded, and/or new materiallayers/elements are included. For example, certain elements disclosed inFIG. 2A are also found in the invention disclosed herein (e.g. a base,analyte sensing layer, an analyte modulating layer etc.) while, as shownin FIG. 2B, other elements are not (e.g. separate HSA protein layers,layers comprising a siloxane adhesion promoter etc.). Similarly,embodiments of the invention include layers/elements having materialsdisposed in unique configurations that are not found in conventionalsensors (e.g. high-density amine (HDA) polymer layers 500).

The conventional embodiment shown in FIG. 2A includes a base layer 102to support the sensor 100. The base layer 102 can be made of a materialsuch as a metal and/or a ceramic and/or a polymeric substrate, which maybe self-supporting or further supported by another material as is knownin the art. Embodiments can include a conductive layer 104 which isdisposed on and/or combined with the base layer 102. Typically, theconductive layer 104 comprises one or more electrically conductiveelements that function as electrodes. An operating sensor 100 typicallyincludes a plurality of electrodes such as a working electrode, acounter electrode and a reference electrode. Other embodiments may alsoinclude a plurality of working and/or counter and/or referenceelectrodes and/or one or more electrodes that performs multiplefunctions, for example one that functions as both as a reference and acounter electrode.

As discussed in detail below, the base layer 102 and/or conductive layer104 can be generated using many known techniques and materials. Incertain embodiments of the invention, the electrical circuit of thesensor is defined by etching the disposed conductive layer 104 into adesired pattern of conductive paths. A typical electrical circuit forthe sensor 100 comprises two or more adjacent conductive paths withregions at a proximal end to form contact pads and regions at a distalend to form sensor electrodes. An electrically insulating cover layer106 such as a polymer coating can be disposed on portions of the sensor100. Acceptable polymer coatings for use as the insulating protectivecover layer 106 can include, but are not limited to, non-toxicbiocompatible polymers such as silicone compounds, polyimides,biocompatible solder masks, epoxy acrylate copolymers, or the like. Inthe sensors of the present invention, one or more exposed regions orapertures 108 can be made through the cover layer 106 to open theconductive layer 104 to the external environment and to, for example,allow an analyte such as glucose to permeate the layers of the sensorand be sensed by the sensing elements. Apertures 108 can be formed by anumber of techniques, including laser ablation, tape masking, chemicalmilling or etching or photolithographic development or the like. Incertain embodiments of the invention, during manufacture, a secondaryphotoresist can also be applied to the protective layer 106 to definethe regions of the protective layer to be removed to form theaperture(s) 108. The exposed electrodes and/or contact pads can alsoundergo secondary processing (e.g. through the apertures 108), such asadditional plating processing, to prepare the surfaces and/or strengthenthe conductive regions.

In the conventional sensor configuration shown in FIG. 2A, an analytesensing layer 110 is disposed on one or more of the exposed electrodesof the conductive layer 104. Typically, the analyte sensing layer 110 isan enzyme layer. Most typically, the analyte sensing layer 110 comprisesan enzyme capable of producing and/or utilizing oxygen and/or hydrogenperoxide, for example the enzyme glucose oxidase. Optionally the enzymein the analyte sensing layer is combined with a second carrier proteinsuch as human serum albumin, bovine serum albumin or the like. In anillustrative embodiment, an oxidoreductase enzyme such as glucoseoxidase in the analyte sensing layer 110 reacts with glucose to producehydrogen peroxide, a compound which then modulates a current at anelectrode. As this modulation of current depends on the concentration ofhydrogen peroxide, and the concentration of hydrogen peroxide correlatesto the concentration of glucose, the concentration of glucose can bedetermined by monitoring this modulation in the current. In a specificembodiment of the invention, the hydrogen peroxide is oxidized at aworking electrode which is an anode (also termed herein the anodicworking electrode), with the resulting current being proportional to thehydrogen peroxide concentration. Such modulations in the current causedby changing hydrogen peroxide concentrations can by monitored by any oneof a variety of sensor detector apparatuses such as a universal sensoramperometric biosensor detector or one of the other variety of similardevices known in the art such as glucose monitoring devices produced byMedtronic Diabetes.

In embodiments of the invention, the analyte sensing layer 110 can beapplied over portions of the conductive layer or over the entire regionof the conductive layer. Typically, the analyte sensing layer 110 isdisposed on the working electrode which can be the anode or the cathode.Optionally, the analyte sensing layer 110 is also disposed on a counterand/or reference electrode. Methods for generating a thin analytesensing layer 110 include brushing the layer onto a substrate (e.g. thereactive surface of a platinum black electrode), as well as spin coatingprocesses, dip and dry processes, low shear spraying processes, ink-jetprinting processes, silk screen processes and the like.

In this context, a variety of pin coating materials and methods areknown in the art (see, e.g. Sahu et al., Indian J. Phys. 83 (4) 493-502(2009), and U.S. Patent Publications 20020127878, 20020127878,20090285982 and 20140272704). In certain embodiments of the invention,the material of the high-density amine layer comprising polymers havinga plurality of repeating amine groups (e.g. poly-l-lysine polymers) isblended with another material such as a solvent or other agent thatmodulates solution viscosity in order to optimize spin coatinguniformity. In this context, to prepare an HDA layer for spin coating,one can mix a viscosity modulating agent and/or one or two or moresolvents together. For example, with two solvents one can use a majorcomponent of something that evaporates relatively quickly and a minorcomponent of something that is relatively slow to evaporate. By usingthis combination, it is often possible to optimize aspects of thisprocess in that during the spin coating process the major componentevaporates quickly to give good coverage and a uniform thick film, andthe remaining minor component still leaves enough plasticity for themolecules to organize before the film is completely dry.

The analyte modulating membrane layer 112 can comprise a glucoselimiting membrane, which regulates the amount of glucose that contactsan enzyme such as glucose oxidase that is present in the analyte sensinglayer. Such glucose limiting membranes can be made from a wide varietyof materials known to be suitable for such purposes, e.g., siliconecompounds such as polydimethyl siloxanes, polyurethanes, polyureacellulose acetates, Nafion, polyester sulfonic acids (e.g. Kodak AQ),hydrogels or any other suitable hydrophilic membranes known to thoseskilled in the art.

In typical embodiments of the invention, a layer of materials comprisinga high-density amine composition layer 500 is disposed between theanalyte modulating layer 112 and the analyte sensing layer 110 as shownin FIG. 2B in order to facilitate their contact and/or adhesion. Intypical embodiments of the invention, the high-density amine layer 500comprises a first side in direct contact with the analyte sensing layer,and a second side in direct contact with the analyte modulating layerand functions as an adhesive layer that binds the analyte sensing layerto the analyte modulating layer. Optionally, the analyte sensing layercomprises glucose oxidase disposed in the layer so that the analytesensor senses glucose; and the high-density amine layer 500 furtherfunctions to decrease sensor signal changes that result from fluctuatinglevels of oxygen (O₂). In certain embodiments of the invention, thepoly-l-lysine in the high-density amine layer 500 has molecular weightsbetween 30 KDa and 300 KDa, for example, molecular weights between 150KDa and 300 KDa. Typically, the poly-l-lysine in the high-density aminelayer 500 is in amounts from 0.1 weight-to-weight percent to 0.5weight-to-weight percent. Optionally, the high-density amine layer 500is from 0.1 to 0.4 microns thick.

Typical Analyte Sensor Constituents of the Invention

The following disclosure provides examples of typicalelements/constituents used in sensor embodiments of the invention. Whilethese elements can be described as discreet units (e.g. layers), thoseof skill in the art understand that sensors can be designed to containelements having a combination of some or all of the material propertiesand/or functions of the elements/constituents discussed below (e.g. anelement that serves both as a supporting base constituent and/or aconductive constituent and/or a matrix for the analyte sensingconstituent and which further functions as an electrode in the sensor).Those in the art understand that selected elements from these thin filmanalyte sensors can be adapted for use in a number of sensor systemssuch as those described herein.

Base Constituent

Sensors of the invention typically include a base constituent (see, e.g.element 102 in FIG. 2A). The term “base constituent” is used hereinaccording to art accepted terminology and refers to the constituent inthe apparatus that typically provides a supporting matrix for theplurality of constituents that are stacked on top of one another andcomprise the functioning sensor. In one form, the base constituentcomprises a thin film sheet of insulative (e.g. electrically insulativeand/or water impermeable) material. This base constituent can be made ofa wide variety of materials having desirable qualities such asdielectric properties, water impermeability and hermeticity. Somematerials include metallic, and/or ceramic and/or polymeric substratesor the like.

Conductive Constituent

The electrochemical sensors of the invention typically include aconductive constituent disposed upon the base constituent that includesat least one electrode for contacting an analyte or its byproduct (e.g.oxygen and/or hydrogen peroxide) to be assayed (see, e.g. element 104 inFIG. 2A). The term “conductive constituent” is used herein according toart accepted terminology. An illustrative example of this is aconductive constituent that forms a working electrode that can measurean increase or decrease in current in response to exposure to a stimulisuch as the change in the concentration of an analyte or its byproductas compared to a reference electrode that does not experience the changein the concentration of the analyte, a coreactant (e.g. oxygen) usedwhen the analyte interacts with a composition (e.g. the enzyme glucoseoxidase) present in analyte sensing constituent 110 or a reactionproduct of this interaction (e.g. hydrogen peroxide). Illustrativeexamples of such elements include electrodes which are capable ofproducing variable detectable signals in the presence of variableconcentrations of molecules such as hydrogen peroxide or oxygen.

In addition to the working electrode, the analyte sensors of theinvention typically include a reference electrode or a combinedreference and counter electrode (also termed a quasi-reference electrodeor a counter/reference electrode). If the sensor does not have acounter/reference electrode then it may include a separate counterelectrode, which may be made from the same or different materials as theworking electrode. Typical sensors of the present invention have one ormore working electrodes and one or more counter, reference, and/orcounter/reference electrodes. One embodiment of the sensor of thepresent invention has two, three or four or more working electrodes.These working electrodes in the sensor may be integrally connected orthey may be kept separate. Optionally, the electrodes can be disposed ona single surface or side of the sensor structure. Alternatively, theelectrodes can be disposed on a multiple surfaces or sides of the sensorstructure (and can for example be connected by vias through the sensormaterial(s) to the surfaces on which the electrodes are disposed). Incertain embodiments of the invention, the reactive surfaces of theelectrodes are of different relative areas/sizes, for example a 1Xreference electrode, a 2.6X working electrode and a 3.6X counterelectrode.

Analyte Sensing Constituent

The electrochemical sensors of the invention include an analyte sensingconstituent disposed on the electrodes of the sensor (see, e.g. element110 in FIG. 2A). The term “analyte sensing constituent” is used hereinaccording to art accepted terminology and refers to a constituentcomprising a material that is capable of recognizing or reacting with ananalyte whose presence is to be detected by the analyte sensorapparatus. Typically, this material in the analyte sensing constituentproduces a detectable signal after interacting with the analyte to besensed, typically via the electrodes of the conductive constituent. Inthis regard, the analyte sensing constituent and the electrodes of theconductive constituent work in combination to produce the electricalsignal that is read by an apparatus associated with the analyte sensor.Typically, the analyte sensing constituent comprises an oxidoreductaseenzyme capable of reacting with and/or producing a molecule whose changein concentration can be measured by measuring the change in the currentat an electrode of the conductive constituent (e.g. oxygen and/orhydrogen peroxide), for example the enzyme glucose oxidase. An enzymecapable of producing a molecule such as hydrogen peroxide can bedisposed on the electrodes according to a number of processes known inthe art. The analyte sensing constituent can coat all or a portion ofthe various electrodes of the sensor. In this context, the analytesensing constituent may coat the electrodes to an equivalent degree.Alternatively, the analyte sensing constituent may coat differentelectrodes to different degrees, with for example the coated surface ofthe working electrode being larger than the coated surface of thecounter and/or reference electrode.

Some sensor embodiments of this element of the invention utilize anenzyme (e.g. glucose oxidase) that optionally has been combined with asecond protein (e.g. albumin) in a fixed ratio (e.g. one that istypically optimized for glucose oxidase stabilizing properties) and thenapplied on the surface of an electrode to form a thin enzymeconstituent. In a typical embodiment, the analyte sensing constituentcomprises a GOx and HSA mixture. In a typical embodiment of an analytesensing constituent having GOx, the GOx reacts with glucose present inthe sensing environment (e.g. the body of a mammal) and generateshydrogen peroxide.

As noted above, the enzyme and the second protein (e.g. an albumin) canbe treated to form a crosslinked matrix (e.g. by adding a cross-linkingagent to the protein mixture). As is known in the art, crosslinkingconditions may be manipulated to modulate factors such as the retainedbiological activity of the enzyme, its mechanical and/or operationalstability. Illustrative crosslinking procedures are described in U.S.patent application Ser. No. 10/335,506 and PCT publication WO 03/035891which are incorporated herein by reference. For example, an aminecross-linking reagent, such as, but not limited to, glutaraldehyde, canbe added to the protein mixture (however in certain embodiments of theinvention disclosed herein, glutaraldehyde is excluded because theaddition of a cross-linking reagent to the protein mixture creates aless active protein paste).

Alternative embodiments of analyte sensing constituents are not formedusing glutaraldehyde, and are instead formed to include entrapped and/orcrosslinked polypeptides such as glucose oxidase crosslinked topolyvinyl alcohol (PVA, see, e.g. CAS number 9002-89-5) polymers. As isknown in the art, polyvinyl alcohol reacts with aldehydes to form waterinsoluble polyacetals. In a pure PVA medium having a pH around 5.0,polymer reaction with dialdehydes is expected to form an acetalcross-linked structure. In certain embodiments of the invention, suchcrosslinking reactions can be performed using a chemical vapordeposition (CVD) process. Due to the acidity of the PVA polymersolution, crosslinking reactions in CVD systems are simple and routine.Moreover, acidic conditions can be created by introducing compounds suchas acetic acid into glutaraldehyde solutions, so a CVD system canprovide an acid vapor condition. In addition the pH of the polymermedium can be adjusted by adding acidic compounds such as citric acid,polymer additives such as polylysine, HBr and the like.

Embodiments of the analyte sensing constituents include compositionshaving properties that make them particularly well suited for use inambulatory glucose sensors of the type worn by diabetic individuals.Such embodiments of the invention include PVA-SbQ compositions for usein layered analyte sensor structures that comprise between 1 mol % and12.5 mol % SbQ. In certain embodiments of the invention that are adaptedor use in glucose sensors, the constituents in this layer are selectedso that the molecular weight of the polyvinyl alcohol is between 30kilodaltons and 150 kilodaltons and the SbQ in the polyvinyl alcohol ispresent in an amount between 1 mol % and 4 mol %. In some embodiments ofthe invention the analyte sensing layer is formed to comprise from 5% to12% PVA by weight. In some embodiments of the invention the analytesensing layer is formed to comprise glucose oxidase in an amount from 10KU/mL to 20 KU/mL.

Embodiments of the analyte sensing constituents include analyte sensinglayers selected for their ability to provide desirable characteristicsfor implantable sensors. In certain embodiments of the invention anamount or ratio of PVA within the composition is used to modulate thewater adsorption of the composition, the crosslinking density of thecomposition etc. Such formulations can readily be evaluated for theireffects on phenomena such as H₂O adsorption, sensor isig drift and invivo start up profiles. Sufficient H₂O adsorption can help to maintain anormal chemical and electrochemical reaction within amperometric analytesensors. Consequently, it is desirable to form such sensors fromcompositions having an appropriate hydrophilic chemistry. In thiscontext, the PVA-GOx compositions disclosed herein can be used to createelectrolyte hydrogels that are useful in internal coating/membranelayers and can also be coated on top of an analyte modulating layer(e.g. a glucose limiting membrane or “GLM”) in order to improve thebiocompatibility and hydrophilicity of the GLM layer.

As noted above, in some embodiments of the invention, the analytesensing constituent includes an agent (e.g. glucose oxidase) capable ofproducing a signal (e.g. a change in oxygen and/or hydrogen peroxideconcentrations) that can be sensed by the electrically conductiveelements (e.g. electrodes which sense changes in oxygen and/or hydrogenperoxide concentrations). However, other useful analyte sensingconstituents can be formed from any composition that is capable ofproducing a detectable signal that can be sensed by the electricallyconductive elements after interacting with a target analyte whosepresence is to be detected. In some embodiments, the compositioncomprises an enzyme that modulates hydrogen peroxide concentrations uponreaction with an analyte to be sensed. Alternatively, the compositioncomprises an enzyme that modulates oxygen concentrations upon reactionwith an analyte to be sensed. In this context, a wide variety of enzymesthat either use or produce hydrogen peroxide and/or oxygen in a reactionwith a physiological analyte are known in the art and these enzymes canbe readily incorporated into the analyte sensing constituentcomposition. A variety of other enzymes known in the art can produceand/or utilize compounds whose modulation can be detected byelectrically conductive elements such as the electrodes that areincorporated into the sensor designs described herein. Such enzymesinclude for example, enzymes specifically described in Table 1, pages15-29 and/or Table 18, pages 111-112 of Protein Immobilization:Fundamentals and Applications (Bioprocess Technology, Vol 14) by RichardF. Taylor (Editor) Publisher: Marcel Dekker; Jan. 7, 1991) the entirecontents of which are incorporated herein by reference.

High-Density Amine Constituent

The electrochemical sensors of the invention include one or morehigh-density amine constituent layers (see, e.g. element 500 in FIG. 2B)that provide the sensors with a number of beneficial functions. Suchlayers can optimize sensor function, for example by acting as anadhesion promoting constituent for layers adjacent to the HDA layer, bydecreasing fluctuations that can occur in glucose oxidase based sensorsin the presence of fluctuating concentration of oxygen, by improvingsensor initialization profiles and the like. The term “adhesionpromoting constituent” is used herein according to art acceptedterminology and refers to a constituent that includes materials selectedfor their ability to promote adhesion between adjoining constituents inthe sensor. Typically, the high-density amine adhesion promotingconstituent is disposed between and in direct contact with the analytesensing constituent and the analyte modulating constituent. In typicalembodiments, the high-density amine layer 500 comprises poly-l-lysinehaving molecular weights between 30 KDa and 300 KDa (e.g. between 150KDa and 300 KDa). The concentrations of poly-l-lysine in suchhigh-density amine layers 500 is typically from 0.1 weight-to-weightpercent to 0.5 weight-to-weight percent and the high-density amine layer500 is from 0.1 to 0.4 microns thick. In embodiments where the analytesensing layer comprises glucose oxidase so that the analyte sensorsenses glucose, and the high-density amine layer 500 functions todecrease sensor signal changes that result from fluctuating levels ofoxygen (O₂).

Analyte Modulating Constituent

The electrochemical sensors of the invention include an analytemodulating constituent disposed on the sensor (see, e.g. element 112 inFIG. 2A). The term “analyte modulating constituent” is used hereinaccording to art accepted terminology and refers to a constituent thattypically forms a membrane on the sensor that operates to modulate thediffusion of one or more analytes, such as glucose, through theconstituent. In certain embodiments of the invention, the analytemodulating constituent is an analyte-limiting membrane which operates toprevent or restrict the diffusion of one or more analytes, such asglucose, through the constituents. In other embodiments of theinvention, the analyte-modulating constituent operates to facilitate thediffusion of one or more analytes, through the constituents. Optionallysuch analyte modulating constituents can be formed to prevent orrestrict the diffusion of one type of molecule through the constituent(e.g. glucose), while at the same time allowing or even facilitating thediffusion of other types of molecules through the constituent (e.g. O₂).

With respect to glucose sensors, in known enzyme electrodes, glucose andoxygen from blood, as well as some interferants, such as ascorbic acidand uric acid, diffuse through a primary membrane of the sensor. As theglucose, oxygen and interferants reach the analyte sensing constituent,an enzyme, such as glucose oxidase, catalyzes the conversion of glucoseto hydrogen peroxide and gluconolactone. The hydrogen peroxide maydiffuse back through the analyte modulating constituent, or it maydiffuse to an electrode where it can be reacted to form oxygen and aproton to produce a current that is proportional to the glucoseconcentration. The analyte modulating sensor membrane assembly servesseveral functions, including selectively allowing the passage of glucosetherethrough (see, e.g. U.S. Patent Application No. 2011-0152654).

C. Typical Analyte Sensor System Embodiments of the Invention

Embodiments of the sensor elements and sensors can be operativelycoupled to a variety of other system elements typically used withanalyte sensors (e.g. structural elements such as piercing members,insertion sets and the like as well as electronic components such asprocessors, monitors, medication infusion pumps and the like), forexample to adapt them for use in various contexts (e.g. implantationwithin a mammal). One embodiment of the invention includes a method ofmonitoring a physiological characteristic of a user using an embodimentof the invention that includes an input element capable of receiving asignal from a sensor that is based on a sensed physiologicalcharacteristic value of the user, and a processor for analyzing thereceived signal. In typical embodiments of the invention, the processordetermines a dynamic behavior of the physiological characteristic valueand provides an observable indicator based upon the dynamic behavior ofthe physiological characteristic value so determined. In someembodiments, the physiological characteristic value is a measure of theconcentration of blood glucose in the user. In other embodiments, theprocess of analyzing the received signal and determining a dynamicbehavior includes repeatedly measuring the physiological characteristicvalue to obtain a series of physiological characteristic values in orderto, for example, incorporate comparative redundancies into a sensorapparatus in a manner designed to provide confirmatory information onsensor function, analyte concentration measurements, the presence ofinterferences and the like.

FIG. 4 shows a schematic of a potentiostat that may be used to measurecurrent in embodiments of the present invention. As shown in FIG. 4, apotentiostat 300 may include an op amp 310 that is connected in anelectrical circuit so as to have two inputs: Vset and Vmeasured. Asshown, Vmeasured is the measured value of the voltage between areference electrode and a working electrode. Vset, on the other hand, isthe optimally desired voltage across the working and referenceelectrodes. The current between the counter and reference electrode ismeasured, creating a current measurement (isig) that is output from thepotentiostat.

Embodiments of the invention include devices which process display datafrom measurements of a sensed physiological characteristic (e.g. bloodglucose concentrations) in a manner and format tailored to allow a userof the device to easily monitor and, if necessary, modulate thephysiological status of that characteristic (e.g. modulation of bloodglucose concentrations via insulin administration). An illustrativeembodiment of the invention is a device comprising a sensor inputcapable of receiving a signal from a sensor, the signal being based on asensed physiological characteristic value of a user; a memory forstoring a plurality of measurements of the sensed physiologicalcharacteristic value of the user from the received signal from thesensor; and a display for presenting a text and/or graphicalrepresentation of the plurality of measurements of the sensedphysiological characteristic value (e.g. text, a line graph or the like,a bar graph or the like, a grid pattern or the like or a combinationthereof). Typically, the graphical representation displays real timemeasurements of the sensed physiological characteristic value. Suchdevices can be used in a variety of contexts, for example in combinationwith other medical apparatuses. In some embodiments of the invention,the device is used in combination with at least one other medical device(e.g. a glucose sensor).

An illustrative system embodiment consists of a glucose sensor, atransmitter and pump receiver and a glucose meter. In this system, radiosignals from the transmitter can be sent to the pump receiver every 5minutes to provide providing real-time sensor glucose (SG) values.Values/graphs are displayed on a monitor of the pump receiver so that auser can self monitor blood glucose and deliver insulin using their owninsulin pump. Typically, an embodiment of device disclosed hereincommunicates with a second medical device via a wired or wirelessconnection. Wireless communication can include for example the receptionof emitted radiation signals as occurs with the transmission of signalsvia RF telemetry, infrared transmissions, optical transmission, sonicand ultrasonic transmissions and the like. Optionally, the device is anintegral part of a medication infusion pump (e.g. an insulin pump).Typically, in such devices, the physiological characteristic valuesinclude a plurality of measurements of blood glucose.

FIG. 3 provides a perspective view of one generalized embodiment ofsubcutaneous sensor insertion system and a block diagram of a sensorelectronics device according to one illustrative embodiment of theinvention. Additional elements typically used with such sensor systemembodiments are disclosed for example in U.S. Patent Application No.20070163894, the contents of which are incorporated by reference. FIG. 3provides a perspective view of a telemetered characteristic monitorsystem 1, including a subcutaneous sensor set 10 provided forsubcutaneous placement of an active portion of a flexible sensor 12, orthe like, at a selected site in the body of a user. The subcutaneous orpercutaneous portion of the sensor set 10 includes a hollow, slottedinsertion needle 14 having a sharpened tip 44, and a cannula 16. Insidethe cannula 16 is a sensing portion 18 of the sensor 12 to expose one ormore sensor electrodes 20 to the user's bodily fluids through a window22 formed in the cannula 16. The sensing portion 18 is joined to aconnection portion 24 that terminates in conductive contact pads, or thelike, which are also exposed through one of the insulative layers. Theconnection portion 24 and the contact pads are generally adapted for adirect wired electrical connection to a suitable monitor 200 coupled toa display 214 for monitoring a user's condition in response to signalsderived from the sensor electrodes 20. The connection portion 24 may beconveniently connected electrically to the monitor 200 or acharacteristic monitor transmitter 100 by a connector block 28 (or thelike).

As shown in FIG. 3, in accordance with embodiments of the presentinvention, subcutaneous sensor set 10 may be configured or formed towork with either a wired or a wireless characteristic monitor system.The proximal part of the sensor 12 is mounted in a mounting base 30adapted for placement onto the skin of a user. The mounting base 30 canbe a pad having an underside surface coated with a suitable pressuresensitive adhesive layer 32, with a peel-off paper strip 34 normallyprovided to cover and protect the adhesive layer 32, until the sensorset 10 is ready for use. The mounting base 30 includes upper and lowerlayers 36 and 38, with the connection portion 24 of the flexible sensor12 being sandwiched between the layers 36 and 38. The connection portion24 has a forward section joined to the active sensing portion 18 of thesensor 12, which is folded angularly to extend downwardly through a bore40 formed in the lower base layer 38. Optionally, the adhesive layer 32(or another portion of the apparatus in contact with in vivo tissue)includes an anti-inflammatory agent to reduce an inflammatory responseand/or anti-bacterial agent to reduce the chance of infection. Theinsertion needle 14 is adapted for slide-fit reception through a needleport 42 formed in the upper base layer 36 and through the lower bore 40in the lower base layer 38. After insertion, the insertion needle 14 iswithdrawn to leave the cannula 16 with the sensing portion 18 and thesensor electrodes 20 in place at the selected insertion site. In thisembodiment, the telemetered characteristic monitor transmitter 100 iscoupled to a sensor set 10 by a cable 102 through a connector 104 thatis electrically coupled to the connector block 28 of the connectorportion 24 of the sensor set 10.

In the embodiment shown in FIG. 3, the telemetered characteristicmonitor 100 includes a housing 106 that supports a printed circuit board108, batteries 110, antenna 112, and the cable 102 with the connector104. In some embodiments, the housing 106 is formed from an upper case114 and a lower case 116 that are sealed with an ultrasonic weld to forma waterproof (or resistant) seal to permit cleaning by immersion (orswabbing) with water, cleaners, alcohol or the like. In someembodiments, the upper and lower case 114 and 116 are formed from amedical grade plastic. However, in alternative embodiments, the uppercase 114 and lower case 116 may be connected together by other methods,such as snap fits, sealing rings, RTV (silicone sealant) and bondedtogether, or the like, or formed from other materials, such as metal,composites, ceramics, or the like. In other embodiments, the separatecase can be eliminated and the assembly is simply potted in epoxy orother moldable materials that is compatible with the electronics andreasonably moisture resistant. As shown, the lower case 116 may have anunderside surface coated with a suitable pressure sensitive adhesivelayer 118, with a peel-off paper strip 120 normally provided to coverand protect the adhesive layer 118, until the sensor set telemeteredcharacteristic monitor transmitter 100 is ready for use.

In the illustrative embodiment shown in FIG. 3, the subcutaneous sensorset 10 facilitates accurate placement of a flexible thin filmelectrochemical sensor 12 of the type used for monitoring specific bloodparameters representative of a user's condition. The sensor 12 monitorsglucose levels in the body, and may be used in conjunction withautomated or semi-automated medication infusion pumps of the external orimplantable type as described in U.S. Pat. Nos. 4,562,751; 4,678,408;4,685,903 or 4,573,994, to control delivery of insulin to a diabeticpatient.

In the illustrative embodiment shown in FIG. 3, the sensor electrodes 10may be used in a variety of sensing applications and may be configuredin a variety of ways. For example, the sensor electrodes 10 may be usedin physiological parameter sensing applications in which some type ofbiomolecule is used as a catalytic agent. For example, the sensorelectrodes 10 may be used in a glucose and oxygen sensor having aglucose oxidase enzyme catalyzing a reaction with the sensor electrodes20. The sensor electrodes 10, along with a biomolecule or some othercatalytic agent, may be placed in a human body in a vascular ornon-vascular environment. For example, the sensor electrodes 20 andbiomolecule may be placed in a vein and be subjected to a blood stream,or may be placed in a subcutaneous or peritoneal region of the humanbody.

In the embodiment of the invention shown in FIG. 3, the monitor ofsensor signals 200 may also be referred to as a sensor electronicsdevice 200. The monitor 200 may include a power source, a sensorinterface, processing electronics (i.e. a processor), and dataformatting electronics. The monitor 200 may be coupled to the sensor set10 by a cable 102 through a connector that is electrically coupled tothe connector block 28 of the connection portion 24. In an alternativeembodiment, the cable may be omitted. In this embodiment of theinvention, the monitor 200 may include an appropriate connector fordirect connection to the connection portion 104 of the sensor set 10.The sensor set 10 may be modified to have the connector portion 104positioned at a different location, e.g., on top of the sensor set tofacilitate placement of the monitor 200 over the sensor set.

While the analyte sensor and sensor systems disclosed herein aretypically designed to be implantable within the body of a mammal, theinventions disclosed herein are not limited to any particularenvironment and can instead be used in a wide variety of contexts, forexample for the analysis of most in vivo and in vitro liquid samplesincluding biological fluids such as interstitial fluids, whole-blood,lymph, plasma, serum, saliva, urine, stool, perspiration, mucus, tears,cerebrospinal fluid, nasal secretion, cervical or vaginal secretion,semen, pleural fluid, amniotic fluid, peritoneal fluid, middle earfluid, joint fluid, gastric aspirate or the like. In addition, solid ordesiccated samples may be dissolved in an appropriate solvent to providea liquid mixture suitable for analysis.

It is to be understood that this invention is not limited to theparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims. In the description of the preferredembodiment, reference is made to the accompanying drawings which form apart hereof, and in which is shown by way of illustration a specificembodiment in which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the present invention.

1. An electrochemical analyte sensor comprising: a base layer; a workingelectrode disposed on the base layer; and a multilayer analyte sensorstack disposed upon the working electrode comprising: (a) an analytesensing layer disposed directly on the working electrode, wherein theanalyte sensing layer detectably alters the electrical current at theworking electrode in the presence of an analyte; (b) a high-densityamine layer disposed over the analyte sensing layer, wherein thehigh-density amine layer comprises polymers having repeating aminegroups; and (c) an analyte modulating layer disposed over thehigh-density amine layer, wherein the analyte modulating layer modulatesthe diffusion of analyte from an external environment to the workingelectrode; wherein: the high-density amine layer comprises a first sidein direct contact with the analyte sensing layer or the analytemodulating layer.
 2. The electrochemical analyte sensor of claim 1,wherein the multilayer analyte sensor stack does not comprise at leastone of: a further layer comprising an albumin; a further layercomprising a siloxane adhesion promoting agent; or a layer comprisingglutaraldehyde.
 3. The electrochemical analyte sensor of claim 1,wherein the analyte sensing layer comprises glucose oxidase.
 4. Theelectrochemical analyte sensor of claim 1, wherein the multilayeranalyte sensor stack consists essentially of (a)-(c).
 5. Theelectrochemical analyte sensor of claim 1, wherein the high-densityamine layer functions as an adhesive layer that binds the analytesensing layer to the analyte modulating layer.
 6. The electrochemicalanalyte sensor of claim 1, wherein the high-density amine layercomprises poly-l-lysine polymers having molecular weights between 30 KDaand 300 KDa.
 7. The electrochemical analyte sensor of claim 6, whereinthe poly-l-lysine in the high-density amine layer has molecular weightsbetween 150 KDa and 300 KDa.
 8. The electrochemical analyte sensor ofclaim 6, wherein the poly-l-lysine in the high-density amine layer is inamounts from 0.1 weight-to-weight percent to 0.5 weight-to-weightpercent.
 9. The electrochemical analyte sensor of claim 1, wherein thehigh-density amine layer is from 0.1 to 0.4 microns thick.
 10. Theelectrochemical analyte sensor of claim 1, wherein the analyte sensinglayer comprises glucose oxidase disposed in the layer so that theanalyte sensor senses glucose; and the high-density amine layerfunctions to decrease sensor signal changes that result from fluctuatinglevels of oxygen (O₂).
 11. A method of making an electrochemical analytesensor comprising the steps of: disposing a working electrode on a baselayer; disposing an analyte sensing layer over the working electrode,wherein the analyte sensing layer detectably alters the electricalcurrent at the working electrode in the presence of an analyte;disposing a high-density amine layer comprising polymers havingrepeating amine groups directly on the analyte sensing layer; anddisposing an analyte modulating layer directly on the high-density aminelayer, wherein: the analyte modulating layer modulates the diffusion ofanalyte therethrough; and the high-density amine layer is formed tocomprise a first side in direct contact with the analyte sensing layeror the analyte modulating layer; so that an electrochemical analytesensor is made.
 12. The method of claim 11, the electrochemical sensorcomprises a multilayer analyte sensor stack disposed over the workingelectrode, said multilayer analyte sensor stack consisting essentiallyof the analyte sensing layer, the high-density amine layer and theanalyte modulating layer.
 13. The method of claim 12, wherein theanalyte sensing layer comprises glucose oxidase disposed in the layer sothat the analyte sensor senses glucose; and the high-density amine layerfunctions to decrease sensor signal changes that result from fluctuatinglevels of oxygen (O₂) during glucose sensing.
 14. The method of claim11, wherein the high-density amine layer is deposited using a spraycoating process and the sensor is exposed to an ethylene oxidesterilization process.
 15. The method of claim 14, wherein the polymershaving repeating amine groups comprise the general structure:

R¹=Alkyl functional groups of various chain lengths (linear and/or brandR²=Ketone functional group R³=Nitrogen functional group
 16. A method ofsensing glucose concentrations in a fluid comprising: (a) disposing anelectrochemical glucose sensor in the fluid, wherein the electrochemicalglucose sensor comprises: a base layer; a working electrode disposed onthe base layer; and a multilayer analyte sensor stack comprising: (i) ananalyte sensing layer comprising glucose oxidase disposed over theworking electrode, wherein the analyte sensing layer detectably altersthe electrical current at the working electrode in the presence of ananalyte; (ii) a high-density amine layer comprising poly-l-lysinepolymers, wherein the high-density amine layer is disposed over theanalyte sensing layer; and (d) an analyte modulating layer disposed overthe high-density amine layer, wherein: the analyte modulating layermodulates the diffusion of glucose therethrough; and the high-densityamine layer comprises a first side in direct contact with the analytesensing layer or the analyte modulating layer; (b) monitoringfluctuations in electrical conductivity; and (c) correlating thefluctuations in electrical conductivity with a concentration of glucose;so that glucose concentrations in the fluid are sensed.
 17. The methodof claim 16, wherein the fluid is interstitial fluid.
 18. The method ofclaim 17, wherein the interstitial fluid is in an individual havingdiabetes.
 19. The method of claim 18, wherein the high-density aminelayer functions to decrease sensor signal changes that result fromfluctuating levels of oxygen (O₂) as glucose concentrations in the fluidare sensed.
 20. The method of claim 18, wherein the analyte modulatinglayer comprises a polyurethane/polyurea polymer formed from a mixturecomprising: (a) a diisocyanate; (b) a hydrophilic polymer comprising ahydrophilic diol or hydrophilic diamine; and (c) a siloxane having anamino, hydroxyl or carboxylic acid functional group at a terminus.